One of the principal mechanisms by which cellular responses are mediated by biologically active molecules including growth factors, cytokines, neurotransmitters and hormones, occurs through the transduction of extracellular signals across the membrane via specific cell-surface receptors and the concomitant generation of second messengers. One such second messenger systems is that of cyclic AMP (cAMP). As a second messenger, cAMP acts to amplify the signal from several ligand effectors and is generated through the action of G proteins coupled to adenylyl cyclase. The primary target of the resulting elevated levels of cAMP is the cAMP-dependent protein kinase A or PKA. The signals generated through PKA have been shown to affect many vital cellular functions including energy metabolism, transcription, differentiation and proliferation, neuronal activity, memory, contractility and motility (Beebe, Semin. Cancer Biol., 1994, 5, 285-294). PKA has also been shown to have an extracellular role in blood through the phosphorylation of vitronectin, a protein associated with platelet activation (Shaltiel et al., Mol. Cell. Biochem., 1993, 127-128, 283-291).
Protein kinase A is a tetrameric holoenzyme composed of two molecules of a regulatory subunit, arranged as a dimer, that bind cAMP and two molecules of a catalytic subunit which, upon enzyme activation (cAMP binding), dissociate from the regulatory dimer and act as serine/threonine kinases (McKnight et al., Recent Prog. Horm. Res., 1988, 44, 307-335). Signal transduction through PKA is tightly regulated with a complexity stemming from auto-inhibitory functions such as autophosphorylation and the existence of several isoforms of both the regulatory and catalytic subunits.
Three isoforms of the catalytic subunit of PKA have been isolated to date, C-alpha, C-beta and C-gamma and all have kinase activity for proteins in the cytoplasm and nucleus (Beebe et al., Mol. Endocrinol., 1990, 4, 465-475; Maldonado and Hanks, Nucleic Acids Res., 1988, 16, 8189-8190). It is the C-alpha subunit, however, that has been most investigated because of its elevated expression, over C-beta and C-gamma, in primary and metastatic melanomas (Becker et al., Oncogene, 1990, 5, 1133-1139).
The C-alpha subunit is also the predominant form of catalytic subunit expressed in TCR-triggered cytotoxic T lymphocytes (CTL) (Sugiyama et al., J. Biol. Chem., 1992, 267, 25256-25263). Studies of the specific role of PKA using antisense oligonucleotides targeting the mouse C-alpha subunit, extending from the -9 to the +9 nucleotides of the translation initiation site, revealed that a reduction in the C-alpha subunit reduced basal PKA activity. Cells treated in parallel were also shown to have enhanced antigen-bearing exocytosis of granules as well as enhanced antigen specific-cytotoxicity. In these studies, the levels of .gamma.-interferon mRNA and protein were also reduced (Sugiyama et al., J. Biol. Chem., 1992, 267, 25256-25263). More recently, the same group investigated the role of C-alpha in the TCR-triggered release of interleukin-2. As before, it was demonstrated that antisense oligonucleotides to the mouse C-alpha subunit, extending from the -6 to the +12 nucleotides of the translation initiation site, inhibited basal PKA activity and decreased interleukin-2 secretion (Sugiyama et al., J. Immunol., 1997, 158, 171-179).
The conclusions drawn from both of these studies involve a dual role for the C-alpha subunit in T-cell receptor (TCR)-triggered T-lymphocytes, that being a mediator of protein synthesis-dependent and -independent processes (Sugiyama et al., J. Biol. Chem., 1992, 267, 25256-25263; Sugiyama et al., J. Immunol., 1997, 158, 171-179).
Overexpression of the C-alpha subunit was also shown to cause growth arrest in CHO (chinese hamster ovary) cells transfected with the full-length cDNA (Tortora et al., Int. J. Cancer, 1994, 59, 712-716).
Meinkoth et al. demonstrated that once activated, the catalytic subunit of PKA undergoes translocation to the nucleus where it participates in transcriptional regulation by phosphorylating nuclear factors. It was also demonstrated that microinjection of the endogeneous PKA inhibitor, PKI, blocked cAMP-mediated changes in gene expression by affecting the activity and localization of C-alpha (Meinkoth et al., Mol. Cell. Biochem., 1993, 127-128, 179-186).
C-alpha phosphorylates binding proteins of the CRE/ATF-1 family (Waeber and Habener, Mol. Endocrinol., 1991, 5, 1431-1438) and microinjection of the C-alpha catalytic subunit has been shown to be sufficient to induce transcription of CRE-regulated genes (Riabowol, Biochem. Cell. Biol., 1992, 70, 1064-1072). In conjunction with the studies in CHO cells, these data suggest that overexpression of the C-alpha subunit might induce growth arrest through the phosphorylation of proteins which operate to negatively control cell proliferation.
Recently, a role for PKA was identified whose mechanism operates via a cAMP-independent pathway and involves only the catalytic subunit. Zhong et al. demonstrated that the transcriptional activity of NF-kB is regulated by the association of the catalytic subunit of PKA with IkB (Zhong et al., Cell, 1997, 89, 413-424). NF-kB is a transcription factor which is retained in the cytosol bound to IkB inhibitory molecules. Upon activation by a wide range of stimuli, the complex is degraded and NF-kB is released allowing it to translocate to the nucleus and activate transcription. Within this novel mechanism, PKA catalytic subunit binds the NF-kB-IkB complex and upon stimulation by an NF-kB ligand, phosphorylates the p65 subunit of NF-kB releasing both factors from the IkB.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of C-alpha. To date, strategies aimed at modulating C-alpha subunit function have involved the use of antibodies, molecules that block the overall function of PKA by targeting the regulatory dimers, antisense expression vectors, and antisense oligonucleotides targeting the mouse isoform.
However, these strategies are either non-specific for the C-alpha subunit or are not therapeutically tested, and consequently, there remains a long felt need for additional agents capable of effectively inhibiting C-alpha function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of C-alpha expression. Therefore, the present invention provides compositions and methods for modulating the catalytic C-alpha subunit of protein kinase A (PKA) through antisense technology.